Vibrio cholerae, the causative agent of cholera, survives and persists in aquatic reservoirs that are subject to fluctuating nutrients and stresses. The bacteria have evolved adaptive responses that allow them to precisely react to these changing environmental inputs with fast and fine-tuned outputs. The molecular circuitry of these signaling networks, however, remains to be fully delineated. In order to better predict and control outbreaks in the future, there is a need for understanding how V. cholerae is able to enhance its aquatic persistence. Toward this end, the long-term goal of this research is to define regulatory mechanisms that V. cholerae uses to coordi- nate physiological responses to environmental cues, in particular, the presence of carbon sources like mannitol and fructose. Both sugars are transported into V. cholerae by the phosphotransferase system (PTS), which is a global regulator of gene expression that coordinates carbohydrate availability and bacterial physiology. Conse- quently, fluctuations in available PTS sugars affect the physiology of V. cholerae. The objective of this application is to elucidate the molecular mechanisms of these processes. The central hypothesis of this proposal is that the regulated expression of PTS components affects the expression of genes involved in biofilm formation by mod- ulating protein activity and second messenger concentrations. Biofilm formation enhances bacterial persistence and the hypothesis is based on results from the prior funding period studying regulation of the mannitol trans- porter (MtlA) of the PTS, a known biofilm enhancer. The rationale for this project is that determination of the mechanisms by which V. cholerae adapts its physiology to these environmentally important sugars will provide a framework from which new strategies to combat cholera can be developed. This project will pursue three aims: 1) Identify the mechanism by which expression of the gene encoding MtlA is repressed; 2) Determine MtlA- dependent and -independent mechanisms of PTS sugar-induced biofilm formation; 3) Map the global network of proteins and regulatory RNAs that modulate V. cholerae physiology upon nutrient shifts. Under the first aim, the genetic interaction between two regulators of mtlA will be tested, and additional factors that affect mtlA expres- sion will be identified. For the second aim, proteins that bind MtlA and impact MtlA-mediated biofilm formation will be determined. Additionally, proteins that impact cyclic diguanylate metabolism and mediate fructose-in- duced biofilm synthesis will be identified. Under the third aim, a combination of mass spectrometry and high- throughput sequencing will be used to identify the full suite of proteins and small RNAs associated with shifts of PTS sugars in the environment. The approach is innovative because it focuses on the survival of V. cholerae in fluctuating environments by defining mechanisms through which synthesis of PTS proteins are regulated and comparing the signaling pathways used by different PTS sugars to drive cellular responses related to persis- tence. The proposed research is significant because understanding how environmental fluxes impact biofilm formation will provide better warning signs of potential epidemics, ultimately decreasing cholera disease burden.